linc-HOXA1 is a non-coding RNA that repressesHoxa1 transcription in cisHédia Maamar1, Moran Cabili2, John Rinn2, Arjun Raj...
Xist is a non-coding RNA that regulatestranscription across an entire chromosomeXist RNA
Hybridization of multiple oligonucleotides yieldsfluorescent spots corresponding to single RNAsTarget mRNATarget mRNAMultip...
The Hox clusters harbor several non-coding RNAsphenomena, polycistronism and antisense transcription.Polycistronic cluster...
linc-HOXA1 is a lincRNA expressed in mouse EScells located near Hoxa1exon3cexon3bexon3aexon 1 exon 2Chr611.5 kb90 kb 41 kb...
There are three isoforms of linc-HOXA1 in mouseembryonic stem cellsexon3cexon3bexon3aexon 1 exon 2Isoform 1Isoform 2Isofor...
Expression of the three isoforms of linc-HOXA1was highly correlated in single cellsexon3cexon3bexon3aexon 1 exon 2Isoform ...
linc-HOXA1 and Hoxa1 transcripts both express inmouse embryonic stem cellsMeannumberofRNApercellA Blinc-HOXA1linc-HOXA1tra...
linc-HOXA1 appears to correlate with Hoxa1 at thepopulation level00 65432120linc-HOXA1Hoxa140Number of days of retinoic ac...
But linc-HOXA1 and Hoxa1 RNA anti-correlate atthe single cell level00 40 80 120linc-HOXA1 RNA0 days retinoicacid exposure1...
Treatment with retinoic acid eliminates this anti-correlation00 965432120Hoxa1Number of days of retinoic acid exposure0 40...
We successfully knocked down linc-HOXA1 withboth Isis anti-sense oligonucleotides and siRNANumberofRNAmoleculespercellcont...
But only the antisense oligonucleotides showed aneffect on Hoxa1... why?NumberofRNAmoleculespercellcontrolknockdowncontrol...
We looked to changes to transcription sites afterlinc-HOXA1 knockdownlinc-HOXA1transcription site
Direct transcript visualization reveals that linc-HOXA1 regulates Hoxa1 at the site of transcriptionSupplementary Figure 5...
Direct transcript visualization reveals that linc-HOXA1 regulates Hoxa1 at the site of transcriptionSupplementary Figure 5...
Transcription site knockdown accounts for thediscrepancy between knockdown methodsNumberofRNAmoleculespercellcontrolknockd...
Transcription site knockdown accounts for thediscrepancy between knockdown methodsNumberofRNAmoleculespercellcontrolknockd...
Over-expression of linc-HOXA1 did not affectHoxa1 expression levels40linc-HOXA1 isoforms Hoxa1 mRNANumberoflinc-HOXA1RNAmo...
Over-expression of linc-HOXA1 did not affectHoxa1 expression levelsHoxa1mRNAmoleculesIsoform 10 150 300Hoxa1linc-HOXA11764...
We pulled down proteins associated with linc-HOXA1 and analyzed by mass-spectrometryBiotinylated RNAPAGE MassspectrometryC...
We pulled down proteins associated with linc-HOXA1 and analyzed by mass-spectrometryBiotinylated RNAPAGE MassspectrometryC...
We pulled down PURB (with a Myc tag) and lookedfor linc-HOXA1 RNA by RT-PCRanti-MycPURB-MycRT-PCR500 bpWater(negativecontr...
Knocking down PURB increased Hoxa1 levels andgot rid of the anti-correlationNumberofRNAmoleculespercellPurb siRNAknockdown...
What lessons does linc-HOXA1 hold for lincRNAfunction?linc-HOXA1transcription site
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linc-HOXA1 is a non-coding RNA that represses Hoxa1 in cis

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linc-HOXA1 is a non-coding RNA that represses Hoxa1 in cis

  1. 1. linc-HOXA1 is a non-coding RNA that repressesHoxa1 transcription in cisHédia Maamar1, Moran Cabili2, John Rinn2, Arjun Raj11University of Pennsylvania2Harvard University and Broad InstituteGenes and Development, in press (2013)Hédia Maamar
  2. 2. Xist is a non-coding RNA that regulatestranscription across an entire chromosomeXist RNA
  3. 3. Hybridization of multiple oligonucleotides yieldsfluorescent spots corresponding to single RNAsTarget mRNATarget mRNAMultiple singly-labeled DNAoligonucleotides, 20 bases eachRaj et al. Nature Methods, 2008RNA fluorescence in situ hybridization(RNA FISH)Femino et al. Science 1998
  4. 4. The Hox clusters harbor several non-coding RNAsphenomena, polycistronism and antisense transcription.Polycistronic clustersA polycistronic cluster designates two or more genes co-transcribed from a single promoter, sharing a non-coding exon,and whose products are generated by alternative splicing [18]. Anoperon is a particular case where the mRNA retains the differentproducts after splicing. In Mammals, both operons andpolycistronic clusters are scarcely documented [18]. One clearexample nonetheless, is the case of the Hoxc4, Hoxc5 and Hoxc6Widespread antisense transcriptionOur analysis also revealed the existence of 15 TUs distinct fromthe Hox and mir genes that are poly-adenylated and alternativelyspliced like genuine products of RNA Polymerase II. Most of theseTUs (14/15) are transcribed antisense (AS) to Hox genes (see fig 2),and AS transcription can represent up to 38% of the splicedtranscripts (38.46% for HoxA, 33.11% for HoxB, 13.16% forHoxC and 34.84% for HoxD). Cis-encoded antisenses andbidirectional promoters are now known to be abundant in theFigure 2. Synopsis of transcriptional activity in the Human Hox Clusters. Sense and antisense transcriptions are in red and green respectively. Darkand light shaded boxes represent exon and intron. Mir genes are in blue. The three long transcripts presenting a murine homolog with a conservedexon-exon boundary are headed by **. The 13 TUs (12 antisense and one sense) located at similar position in human and mouse are denoted by anasterisk (*) while the 10 TUs showing conservation are depicted withdoi:10.1371/journal.pone.0000356.g002Rinn et al. Cell 2007Mainguy et al. PLOS ONE 2007
  5. 5. linc-HOXA1 is a lincRNA expressed in mouse EScells located near Hoxa1exon3cexon3bexon3aexon 1 exon 2Chr611.5 kb90 kb 41 kb 3 kbIsoform 1Isoform 2Isoform 3Skap2 Hoxa1 Hoxa2 Hoxa3linc-HOXA1
  6. 6. There are three isoforms of linc-HOXA1 in mouseembryonic stem cellsexon3cexon3bexon3aexon 1 exon 2Isoform 1Isoform 2Isoform 3Isoform 1Isoform 2Isoform 3exon 3c exon 3b exon 3alinc-HOXA1
  7. 7. Expression of the three isoforms of linc-HOXA1was highly correlated in single cellsexon3cexon3bexon3aexon 1 exon 2Isoform 1Isoform 2Isoform 3Isoform 1Isoform 2Isoform 3exon 3c exon 3b exon 3alinc-HOXA112012090906060E3a molecules per cellE3bmoleculespercellE3cmoleculespercellE3cmoleculespercell303000 1209060E3a molecules per cell300 1209060E3b molecules per cell300y = ax + ba = 1.296 [1.084, 1.507]b = 4.893 [2.568, 7.219]y = ax + ba = 2.031 [1.750, 2.312]b = 9.202 [6.112, 12.29]y = ax + ba = 1.253 [1.095, 1.411]b = 6.652 [3.552, 9.753]
  8. 8. linc-HOXA1 and Hoxa1 transcripts both express inmouse embryonic stem cellsMeannumberofRNApercellA Blinc-HOXA1linc-HOXA1transcription siteHoxa1
  9. 9. linc-HOXA1 appears to correlate with Hoxa1 at thepopulation level00 65432120linc-HOXA1Hoxa140Number of days of retinoic acid expos0 days retinoicacid exposure1 day retinoicacid exposure2 daacidMeannumberofRNApercell180 180ACBlinc-HOXA1linc-HOXA1transcription siteHoxa1Threshold00 965432120linc-HOXA1Hoxa140Number of days of retinoic acid exposure0 days retinoicacid exposure1 day retinoicacid exposure2 days retinoicacid exposureMeannumberofRNApercell4060120180120180Hoxa1mRNAACBlinc-HOXA1linc-HOXA1transcription siteHoxa1Hoxa1linc-HOXA11252612735n = 313, p = 0.33Hoxa1linc-HOXA1261071101n = 235, p = 10-5Hoxa1linc-HOXA125456431n = 354, p = 0.007Threshold
  10. 10. But linc-HOXA1 and Hoxa1 RNA anti-correlate atthe single cell level00 40 80 120linc-HOXA1 RNA0 days retinoicacid exposure1 day retinoacid exposu0 40linc-HOXA1 RMean0204060060120180Hoxa1mRNACnHoxa1linc-HOXA1261071101n = 235, p = 10-5ThresholdThreshold
  11. 11. Treatment with retinoic acid eliminates this anti-correlation00 965432120Hoxa1Number of days of retinoic acid exposure0 40 80 120linc-HOXA1 RNA0 days retinoicacid exposure1 day retinoicacid exposure2 days retinoicacid exposure0 40 80 120linc-HOXA1 RNA0 40 80 120linc-HOXA1 RNAMeannumberofRNAp0204060060120180060120180Hoxa1mRNACHoxa1linc-HOXA11252612735n = 313, p = 0.33Hoxa1linc-HOXA1261071101n = 235, p = 10-5Hoxa1linc-HOXA125456431n = 354, p = 0.0077ThresholdThreshold
  12. 12. We successfully knocked down linc-HOXA1 withboth Isis anti-sense oligonucleotides and siRNANumberofRNAmoleculespercellcontrolknockdowncontrolknockdowncontrolknockdowncontrolknockdown1216Isis knockdown siRNA knockdownlinc-HOXA1knockdownlinc-HOXA1 Hoxa1840linc-HOXA1 Hoxa1
  13. 13. But only the antisense oligonucleotides showed aneffect on Hoxa1... why?NumberofRNAmoleculespercellcontrolknockdowncontrolknockdowncontrolknockdowncontrolknockdown1216Isis knockdown siRNA knockdownlinc-HOXA1knockdownlinc-HOXA1 Hoxa1840linc-HOXA1 Hoxa1
  14. 14. We looked to changes to transcription sites afterlinc-HOXA1 knockdownlinc-HOXA1transcription site
  15. 15. Direct transcript visualization reveals that linc-HOXA1 regulates Hoxa1 at the site of transcriptionSupplementary Figure 5DAPIControl (scramble knockdown) linc-HOXA1 knockdownsiRNAIsisanti-senseoligonucleotidesDAPIlinc-HOXA1 exon linc-HOXA1 exon
  16. 16. Direct transcript visualization reveals that linc-HOXA1 regulates Hoxa1 at the site of transcriptionSupplementary Figure 5DAPIControl (scramble knockdown) linc-HOXA1 knockdownsiRNAIsisanti-senseoligonucleotidesDAPIlinc-HOXA1 exon linc-HOXA1 exon
  17. 17. Transcription site knockdown accounts for thediscrepancy between knockdown methodsNumberofRNAmoleculespercellcontrolknockdowncontrolknockdowncontrolknockdowncontrolknockdown1216Isis knockdown siRNA knockdownlinc-HOXA1knockdownlinc-HOXA1 Hoxa1840linc-HOXA1 Hoxa1
  18. 18. Transcription site knockdown accounts for thediscrepancy between knockdown methodsNumberofRNAmoleculespercellcontrolknockdowncontrolknockdowncontrolknockdowncontrolknockdown1216Isis knockdown siRNA knockdownlinc-HOXA1knockdownlinc-HOXA1 Hoxa1840linc-HOXA1 Hoxa1Number of activetranscriptionsites per cellNumber of activetranscriptionsites per cell0.40.200.20
  19. 19. Over-expression of linc-HOXA1 did not affectHoxa1 expression levels40linc-HOXA1 isoforms Hoxa1 mRNANumberoflinc-HOXA1RNAmolecules60200NumberofHoxa1mRNAmolecules20100GFP(control)Isoform1Isoform2Isoform3GFP(control)Isoform1Isoform2Isoform3
  20. 20. Over-expression of linc-HOXA1 did not affectHoxa1 expression levelsHoxa1mRNAmoleculesIsoform 10 150 300Hoxa1linc-HOXA117641558n = 154, p = 0.95Isoform 20 150 300Hoxa1linc-HOXA18401977n = 144, p = 0.65linc-HOXA1 RNA molecules linc-HOXA1 RNA molecules linc-HOXA1 RNA molecules linc-HOXA1 RNA moleculesIsoform 30 150 300Hoxa1linc-HOXA17242192n = 144, p = 0.6240800GFP (control)0 30 60Hoxa1linc-HOXA12611269n = 153, p = 0.055Threshold
  21. 21. We pulled down proteins associated with linc-HOXA1 and analyzed by mass-spectrometryBiotinylated RNAPAGE MassspectrometryCell lysate
  22. 22. We pulled down proteins associated with linc-HOXA1 and analyzed by mass-spectrometryBiotinylated RNAPAGE MassspectrometryCell lysateexon3canti-senseexon3csenseexon3a,banti-senseexon3a,bsenseexon3asense30405080PURB(mass-spectrometry)exon3cexon3bexon3aexon 1 exon 2linc-HOXA1 GAA-rich region
  23. 23. We pulled down PURB (with a Myc tag) and lookedfor linc-HOXA1 RNA by RT-PCRanti-MycPURB-MycRT-PCR500 bpWater(negativecontrol)TotallysateIgGRNApulldownMYCRNApulldown200 bp
  24. 24. Knocking down PURB increased Hoxa1 levels andgot rid of the anti-correlationNumberofRNAmoleculespercellPurb siRNAknockdownlinc-HOXA1 Hoxa1Purb1525201050controlknockdowncontrolknockdowncontrolknockdownPurb siRNAknockdown0 60Threshold120linc-HOXA1 RNA0204060Hoxa1mRNAHoxa1linc-HOXA1123236n = 44, p = 0.96
  25. 25. What lessons does linc-HOXA1 hold for lincRNAfunction?linc-HOXA1transcription site
  26. 26. Thanks for watching!

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